Site‐directed mutagenesis of Klebsiella aerogenes urease: Identification of histidine residues that appear to function in nickel ligation, substrate binding, and catalysis

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Abstract
Comparison of six urease sequences revealed the presence of 10 conserved histidine residues (H96 in the γ subunit, H39 and H41 in β, and H134, H136, H219, H246, H312, H320, and H321 in the α subunit of the Klebsiella aerogenes enzyme). Each of these residues in K. aerogenes urease was substituted with alanine by site-directed mutagenesis, and the mutant proteins were purified and characterized in order to identify essential histidine residues and assign their roles. The γH96A, βH39A, βH41A, αH312A, and αH321A mutant proteins possess activities and nickel contents similar to wild-type enzyme, suggesting that these residues are not essential for substrate binding, catalysis, or metal binding. In contrast, the αH134A, αH136A, and αH246A proteins exhibit no detectable activity and possess 53%, 6%, and 21% of the nickel content of wild-type enzyme. These results are consistent with αH134, αH136, and αH246 functioning as nickel ligands. The αH219A protein is active and has nickel (∼1.9% and ∼80%, respectively, when compared to wild-type protein) but exhibits a very high Km value (1, 100 + 40 mM compared to 2.3 + 0.2 mM for the wild-type enzyme). These results are compatible with αH219 having some role in facilitating substrate binding. Finally, the αH320A protein (Km = 8.3 + 0.2 mM) only displays ∼0.003% of the wild-type enzyme activity, despite having a normal nickel content. Unlike the wild-type and αH219A ureases, this mutant protein was not inactivated by diethylpyrocarbonate (DEP), consistent with αH320 being the DEP-reactive general base that facilitates catalysis.